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Testing Status of Agents at NTP
Testing Status of Agents at NTP
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1. Human Data
No data were found on the chemical disposition of
TCAB or TCAOB in humans.
2. Animal Data
B. Acute
1. Human Data
No data were found on the acute toxicity of TCAB
or TCAOB in humans.
2. Animal Data
E.I. du Pont de Nemours & Company (Haskell
Laboratories) conducted the following acute toxicity test on laboratory
animals:
Compound Tested | Formulation for Ear Test mg/ml (MIBK)a | TCAB Content of Sample (µg/g) | Total dose TCAB Applied (µg) | Microscopic Evaluation of Rabbit Ear Testb |
---|---|---|---|---|
TCAB | 0.001 | -- | 0.5 | -,+ |
TCAB | 0.01 | -- | 5 | +,++ |
TCAB | 0.02 | -- | 8 | +,++ |
TCAB | 0.04 | -- | 16 | ++,++ |
TCAB | 0.08 | -- | 32 | ++,++ |
NOTES:
- = no hyperkeratosis
+ = mild hyperkeratosis
++ = moderate hyperkeratosis
a) MIBK = methyl isobutylketone
b) Two rabbits per test were evaluated. If both "treated" ears reacted the same, only one rating was given; if they reacted differently, both ratings were given.
___________
Reference: Hill et al., 1981
C. Prechronic
1. Epidemiological Evidence/Case Reports
A number of cases of chloracne have been reported
following occupational exposure to herbicides. In these cases,
the observed chloracne was attributed to TCAB and/or TCAOB. In
general, chloracne is characterized by comedone formation, straw-colored
cysts, and inflammatory papules. The most sensitive area of the
skin is around the eyes and ears. Chloracne may be associated
with systemic toxicity. Direct skin contact is expected to be
the primary route of exposure; however, inhalation and ingestion
are speculative routes.
Seven to eight years later, a long-term follow-up
study was conducted on 5 of the workers and 2 children who had
developed chloracne. Three of the workers still had evidence of
chloracne. Four of the five workers were sensitive to sunlight.
The 2 children had mild scarring, and one of the children (a 15-year-old
girl) had acne vulgaris [Taylor and Lloyd, 1982].
Location | Year | Herbicide | Source | Number of Workers (%) | Probable Chloracnegenic Chemical |
---|---|---|---|---|---|
Illinois | 1960s and/or 1970s | Methazole | Chemical Plant | <10 | TCAOB |
England | 1970s | Methazole | Chemical Plant | U | TCAOB |
Ohio*1 | 1972-73 | Methazole | Chemical Plant | 41 (90%) | TCAOB |
Michigan | 1970s | Methazole | Chemical Plant | U | TCAOB |
New Jersey | 1960s and/or 1970s | Propanil | Chemical Plant | U | TCAB |
Southern U.S.A. | 1960s and/or 1970s | Propanil | Railroad cars delivering herbicide | U | TCAB |
Arkansas**1 | 1974-77 | Propanil | Chemical Plant | 17 (61%) | TCAB |
Europe | 1970s | U | Chemical Palnt | U | TCAB |
U--unknown; *Taylor and co-workers, 1974; **Morse and co-workers, 1977, 1979.
1--Previously described.
___________
Reference: Taylor and Lloyd, 1982
2. Animal Data
In mice and rats, both compounds caused histopathological
changes of the liver, including hypertrophy and hyperplasia (greater
than 50% compared to controls). Liver cells of the TCAB and TCAOB
treated rats had granular cytoplasm containing vacuoles; some
hepatocytes contained mitotic figures (P < 0.001). In addition,
TCAB and TCAOB treatment induced proliferation of the smooth endoplasmic
reticulum of rat liver cells. Mice livers treated with TCAB developed
mitotic figures in which chromosomes appeared in a tripolar arrangement
[Schrankel et al., 1980].
In study 1, male mice from each strain were tested
with 0.001% TCAOB in acetone 5 days per week for 9 weeks. Termination
of the experiment was based on mortality. Three positive control
rabbits were treated with 0.001% TCAB as follows: 1 per week for
17.5 weeks; 3 times per week for 17.5 weeks; or 5 times per week
for 6 weeks.
In study 2, female rhino and hairless mice received
daily applications of 0.001%, 0.01%, and 0.1% TCAOB five days
per week until fatalities occurred. Two positive control male
rabbits received 0.001% TCAOB daily and 100 microliters of acetone.
In study 3, male hairless mice and rhino mice received
doses of 0.01 and 0.1% TCAOB. Rhino mice received 18 treatments
(3.5 weeks) at both dose levels and hairless mice received 12
treatments of 0.1% TCAOB or 18 treatments of 0.01% TCAOB.
Based on gross and histologic examination of the
skin, no abnormalities occurred in treated mice from study 1.
However, signs of chloracne, including hyperplasia and hypertrophy
of the right ear, were seen in rabbits within 2.5 weeks. A dose-dependent
follicular hyperkeratosis and epithelial hyperplasia (characteristic
of a chloracnegenic response) were seen in mice in studies 2 and
3. Mice receiving 0.01% and 0.1% TCAOB experienced more severe
effects including erythema, skin-fold thickening, discoloration,
and weight loss. In studies 2 and 3, eyes of the rhino mice treated
with TCAOB were swollen shut with keratinaceous ocular discharge.
Rabbits also developed chloracne. One male hairless mouse in the
0.1% TCAOB dose group and one rhino mouse in the 0.01% TCAOB dose
group died at 3 and 4 weeks, respectively. Rhino mice in the 0.1%
TCAOB dose group developed necrotic foci of the liver [Horton
and Yeary, 1985].
E.I. du Pont de Nemours & Company, Inc., (Haskell
Laboratories), conducted the following prechronic skin absorption
studies in albino rabbits of unspecified strain:
° In the second part of the study, 40 male
albino rabbits were divided into groups in order to establish
a no-effect level for "Still Bottom Tars." Groups 1
and 2 were divided into 2 dose groups. Group 1a (n=15) (control)
received 3.5 milliliters of acetone for 10 days (wrapped). Group
1b (n=10) received 0.35 milliliters of acetone for 20 days (not
wrapped). Group 2 (n=10) received 0.35 milliliters TCAB in 0.1%
acetone solution (unwrapped) for 20 days. The day after the 10th
treatment, five animals from each group were sacrificed. The remaining
rabbits were sacrificed on day 14 or 30. Blood was taken from
the marginal ear vein before the test began, after the 10th treatment,
after the 30th treatment (group 1 and 2), and 14 days after the
last treatment. By day 6, severe skin irritation had developed
in all test groups with characteristics similar to those described
for the first part of the study. [E.I. du Pont de Nemours &
Company, Inc., 1982f].
TCAB (concentrations ranging from 0.002%-2.0% TCAB
in 2,3-DCA) and DCA were acnegenic. DCA 50% by weight in chloroform
and DCA 50% by weight in 2,3-DCA produced only mild acnegenic
effects accompanied by pore enlargement. Gross examination of
the skin revealed erythema, sloughing, and ear thickening. Pathological
findings included hair follicles filled with yellow plugs and
keratinous material on the skin [E.I. du Pont de Nemours &
Company, Inc, 1982a].
1. Human Data
No data were found on the chronic/carcinogenic effects
of TCAB or TCAOB in humans.
2. Animal Data
A significant decrease in body weight was seen in the TCAB (9.4%) and TCAOB (16.9%) treated rats compared to controls. Hematocrit values and hemoglobin levels decreased in test groups. This decrease was more significant in TCAOB-treated rats (P < 0.001) than TCAB treated rats (P < 0.05). The white blood cell count was insignificantly decreased in both treated groups. The red cell count was significantly decreased in the TCAOB group (P < 0.001) and insignificantly decreased in the TCAB treated group. Liver, spleen, and testicular weights increased significantly (P < 0.05, P < 0.05, and P < 0.005, respectively) in TCAOB treated rats compared to controls. These organ weights were insignificantly increased in TCAB treated rats. Biochemical measurements conducted in this study are reported in section VG.3 [Hsia et al., 1980].
E. Reproductive Effects and Teratogenicity
1. Human Data
No data were found on the reproductive or teratogenic
effects of TCAB or TCAOB in humans.
2. Animal Data
Adverse effects in the treated groups were compared
to controls. The only maternal toxic effect observed was a significant
(P < 0.05) decrease in the thymus weight in the 10 ppm TCAOB
treated group. A significant (P < 0.01) decrease in the number
of pups per female whelping at birth and at weaning was observed
in the 10 ppm TCAOB treated mice. A significant (P < 0.01)
increase in pup weight at birth compared to controls was observed
in the 1 ppm TCAOB treated group. The sum of the individual pup
weights (litter mass) was significantly decreased at birth and
on days 7, 14, 21, and 28 post-partum (P < 0.01, P < 0.05,
P < 0.01, P < 0.01, and P < 0.01, respectively) in
the 10 ppm TCAOB treated group. A significant (P < 0.05) decrease
in the litter mass was also observed in the 1 ppm TCAOB treated
group on day 21.
In the 28-day-old pups used to assess immune function,
thymus weights were insignificantly less than controls. However,
the thymus weights of the 10 ppm TCAOB treated pups not immunized
with SRBCs were significantly (P< 0.01) lower than control
pups. In the immunized mice, no significant difference in liver
and spleen weights was observed compared to control mice. However,
plaque forming cells were significantly decreased (P < 0.01)
compared to control mice [Bleavins et al., 1985a].
In addition, pregnant C57BL mice were treated with
dioxane or TCAOB at 8 mg/kg on day 12, or cortisone acetate at
2.5 mg/animal on days 11-14 and sacrificed on day 15. The embryos
were removed and heads were prepared for electron microscope viewing
in order to examine palate cells.
The specific dosing scheme and results from the experiments
outlined above are presented in Table 5.
As part of the same study, the effect of TCAOB in the offspring of the above matings (AKR X C57BL; C57BL X AKR; NMRI X DBA) was compared to the effect of this compound on inbred parental strains. Backcrosses between the F1 generation of NMRF and DBW with inbred NMRI was also tested. The specific treatment regimen and experimental results are described in Table 6.
Strain | Day Treated (3 p.m.) | Dosage | Dams with malformed fetuses %, (no. of affectedb/treated) | No. of implan- tations | Resorption + dead fetuses % (early/late)c | Cleft Palate %d | Hydro- nephrosis % | Hydrops % |
---|---|---|---|---|---|---|---|---|
C57BL | 10 | TCAOB 6 mg/kg | 100 (11/11) | 77 | 31.2 (15/9) | 33.6 | 63 | 0 |
10 | Dioxanea | 0 (0/7) | 46 | 19.6 (9/0) | 0 | 0 | 0 | |
11 | TCAOB 6mg/kg | 100 (10/10) | 77 | 28.6 (15/7) | 64.7 | 79.3 | 5.1 | |
11 | Dioxanea | 28.6 (2/7) | 52 | 15.4 (7/1) | 4.5 | 0 | 0 | |
12 | TCAOB 6mg/kg | 100 (11/11) | 80 | 21.3 (9/8) | 56.6 | 37.3 | 8.1 | |
12 | TCAOB 16mg/kg | 66.7 (6/9) | 50 | 60 (29/1) | 95 | __ | 2 | |
12 | Dioxanea | 40 (2/5) | 35 | 14.3 (5/0) | 0 | 12.5 | 0 | |
13 | TCAOB 6mg/kg | 90.9 (10/11) | 82 | 19.5 (8/8) | 23 | 26.9 | 7.9 | |
13 | Dioxanea | 0 (0/5) | 33 | 18.2 (6/0) | 0 | 0 | 0 | |
DBA | 10 | TCAOB 8mg/kg | 28.6 (2/7) | 54 | 5.6 (2/1) | 1.9 | 2 | 0 |
11 | TCAOB 8mg/kg | 28.6 (2/7) | 50 | 20 (10/0) | 5 | 0 | 0 | |
12 | TCAOB 8mg/kg | 14.3 (1/7) | 48 | 8.3 (4/0) | 2.3 | 0 | 0 | |
12 | TCAOB 16mg/kg | 11.1 (1/9) | 52 | 38.4 (14/6) | 3.1 | __ | 0 | |
AKR | 10 | TCAOB 8mg/kg | 12.5 (1/8) | 38 | 10.5 (4/0) | 0 | 2.9 | 0 |
11 | TCAOB 8mg/kg | 25 (2/8) | 34 | 14.7 (5/0) | 0 | 10.3 | 0 | |
12 | TCAOB | 28.6 (2/7) | 40 | 7.5 (3/0) | 2.7 | 2.7 | 0 |
a Dioxane used as solvent for the TCAOB
b Malformations only
c Dead Fetuses less than about 6 mm of length have been assigned to the group of early dead
d Based on fetuses being alive or dead in late stage and possible to investigate
___ Not investigated
__________
Reference: Hassoun et al., 1984
No. of live fetuses | Cleft palate % of live fetusesa |
|||||||
---|---|---|---|---|---|---|---|---|
Strain | Dose mg/kg | No. of dams | No. of implan- tations | Resorptions + dead fetuses % | Non- pigm. | Pigm. | Non- pigm. | Pigm. |
1) C57BL | 6 | 11 | 80 | 21.3 | __ | 63 | __ | 50.8 |
2) AKR | 8 | 7 | 40 | 7.5 | 37 | __ | 2.7 | __ |
3) C57BL fx AKR m | 6 | 12 | 84 | 11.9 | __ | 74 | __ | 1.4 |
4) C57BL fx C57BL m | 10 | 10 | 73 | 16.4 | __ | 61 | __ | 1.6 |
5) AKR fx C57BL m | 6 | 9 | 19 | 10.5 | __ | 17 | __ | 0 |
6) (AKR x C57BL) fx AKR m | 10 | 10 | 95 | 4.2 | 47 | 44 | 2.2 | 0 |
7) AKR fx (AKR x C57BL) m | 10 | 10 | 55 | 18.2 | 21 | 24 | 4.8 | 8.3 |
8) NMRI | 8 | 16 | 147 | 8.8 | 134 | __ | 90.3 | __ |
9) DBA | 8 | 7 | 48 | 8.3 | __ | 44 | __ | 2.3 |
10)NMRI fx DBA m | 8 | 12 | 102 | 9.8 | __ | 92 | __ | 6.5 |
11)NMRI fx (NMRI x DBA) m | 8 | 16 | 148 | 9.5 | 66 | 68 | 48.5b | 51.5b |
12)(NMRI x DBA) fx NMRI m | 8 | 17 | 150 | 5.3 | 61 | 80 | 18b | 23.8b |
f =female
m =male
aSee Table 5
bPercent malformations among combined black and white offspring of NMRI fx (NMRI x DBA) m significantly different
from that of (NMRI x DBA) fx NMRI m
P<0.01
__________
Reference: Hossoun et al., 1984
No evidence of maternal toxicity was observed in
either the Ah-responsive or Ah-nonresponsive mice.
C57BL mice treated with 6 mg/kg TCAOB on days 10, 11, 12, or 13
of gestation had an increased percentage of malformed fetuses
(90.9-100%). Malformations observed included cleft palate, hydronophrosis,
and hydrops in several cases (see Table 5). The percentage of
hydrops increased in C57BL mice following treatment with 6 mg/kg
TCAOB on days 11, 12, and 13 of gestation. Treatment of C57BL
mice with 16 mg/kg TCAOB on day 12 of gestation resulted in a
60% occurrence of resorbed and dead fetuses, and a 95% occurrence
of cleft palate. The frequency of the four parameters of fetal
toxicity presented in Table 5 changed with time of administration
in the C57BL dams. Generally, TCAOB treatment resulted in high
frequency of late fetal death. An exception to this was observed
in the group treated with 16 mg/kg TCAOB, where the frequency
of hydrops showed a tendency to increase (not significantly) when
the TCAOB was given late. Treatment of DBA mice with 16 mg/kg
TCAOB caused an increase in resorption and fetal death (38%) compared
to controls; however, no increase in cleft palate was noted. No
evidence of toxicity was seen in the AKR mice after treatment
with 8 mg/kg TCAOB on days 10, 11, or 12 of gestation. The authors
suggested that this increase in sensitivity to TCAOB treatment
by the Ah-responsive mice demonstrates an involvement of
the Ah-locus in cleft palate formation.
The cross breeding study indicated that "the
nonresponsiveness of DBA and AKR mice segregates as a dominant
trait" in the crosses with C57BL and NMRI, respectively.
The authors suggest that the percentage of malformations was significantly
higher among the backcross fetuses where the mother was an inbred
NMRI (father NMRI x DBA) compared to the situation where the mother
was NMRI x DBA (father inbred NMRI). Provided that the sensitivity
is not linked to the sex chromosomes, the authors suggest that
the host maternal factor is involved in the teratogenic mechanism.
NMRI mice had a high incidence of cleft palate formation in the
8.0 mg/kg TCAOB dosed group on day 12 of gestation. The authors
also assert that because approximately 20% of the offspring had
cleft palate after TCAOB treatment, the fetal genotype may be
determined by sensitivity to the teratogenic action of TCAOB.
Examination by scanning electron microscopy of the
palate cells from embryos of pregnant C57BL mice treated on day
14 with TCAOB did not reveal degeneration [Hassoun et al.,
1984].
None of the DBA fetuses (including those transferred
into NMRI uteri) developed cleft palates. Of the NMRI fetuses
that remained in the NMRI uteri following treatment with TCDD,
85% (29/34) had cleft palate while 100% (11/11) of the NMRI fetuses
in the DBA dams had cleft palate. In the TCAOB treatment group,
90% (56/62) of the NMRI fetuses that remained in the NMRI uteri
developed cleft palate and 93% (13/14) developed cleft palate
following transfer to a DBA uterus [D'Argy et al., 1984].
Maternal toxicity from TCAOB or DFMO treatment was not observed. The effects of TCAOB treatment on NMRI pregnant mice on day 11 and DFMO on days 11 through 12 of gestation are presented in Table 7. The effects of the treatment of pregnant NMRI mice with TCAOB on day 12 of gestation and DFMO on days 12 through 13 of gestation are presented in Table 8. Administration of DMFO at any dose produced no cleft palates. At a dose of 300 mg/kg, DMFO increased the rate of fetal death compared to that observed in the vehicle control group (P < 0.02). DFMO decreased the frequency of cleft palate when co-administered with TCAOB. TCAOB-induced fetal death was not affected by DFMO administration. The authors concluded that the possible mechanism of cleft palate formation involves TCAOB stimulation of the polysubstrate monooxygenase system and other enzymes of the Ah-locus system, thus keeping epithelial cells alive leading to the formation of cleft palate. DFMO inhibits the activity of this enzyme and thereby decreases cleft palate formation [Hassoun and Arif, 1988].
Dosage | Dams with Fetuses Having Cleft Palate % and (Number of Affected/Treated) | Mean of Implantation Number/Dam ± S.E.M. and (Total Number) | Percent of Fetuses/Dam Being Resorbed or Dead ± S.E.M. and (Number Early/Number Late)a | Percent of Fetuses/Dam Having Cleft Palate ± S.E.M. and (Number Among Investigated) |
---|---|---|---|---|
No Treatment | 0 (0/11) | 6.1 ± 0.73 (67) | 11.9 &3177; 4.3 (7/1) | 0 (0/60) |
DFMO + dioxane | 0 (0/10) | 8.8 ± 0.56 (88) | 13.6 ± 3.4 (10/0) | 0 (0/78) |
TCAOB + saline | 42.9 (6/14) | 7.6 ± 0.62 (106) | 33.9 ± 8.1* (25/11) | 37.0 ± 7.4 (30/81) |
TCAOB + DFMO | 33.3 (6/18) | 8.9 ± 0.43 (160) | 36.3 ± 10.7 (38/20) | 17.2 ± 3.2** (21/122) |
a Resorbed plus dead fetuses (<6 mm of length) have been assigned to the group of early dead, while dead fetuses more than 6 mm of length have been assigned to the group of late dead.
* Significantly different from control (DMFO-plus dioxane-treated), by Student's t-test, P = 0.025.
** Significantly different from control (saline-plus
TCAOB-treated), by Students's t-test, P < 0.01.
Dosage | Dams with Fetuses Having Cleft Palate % and (Number of Affected/Treated) | Mean of
Implan- tation Number/ Dam ± S.E.M. and (Total Number) | Percent of Fetuses/Dam Being Resorbed or Dead ± S.E.M. and (Number Early/Number Late)a | Percent of Fetuses/ Dam Having Cleft Palate ± S.E.M. and (Number Among Investigated) |
---|---|---|---|---|
DFMO + dioxane | 0 (0/7) | 9.3 ± 0.58 (65) | 9.2 ± 3.1 (6/0) | 0 (0/59) |
TCAOB + saline | 100 (14/14) | 7.6 ± 0.68 (106) | 11.3 ± 4.3 (7/5) | 77.8 ± 8.2 (76/99) |
TCAOB + DFMO | 75 (12/16) | 7.5 ± 0.60 (120) | 13.3 ± 3.4 (16.0) | 42.3 ± 7.5* (44/104) |
* Significantly different from control (saline-plus TCAOB-treated), by Students's t-test, P < 0.005.
__________
Reference: Hassoun and Arif, 1988
An insignificant decrease in hatchability was observed
in eggs injected with TCAB and TCAOB on day 4 compared to controls.
The majority of deaths occurred before day 13 of incubation. Few
deaths were observed in eggs injected on days 11-13. A 100% mortality
was observed in eggs treated with 1.0 µg TCAB and 0.1 µg
TCAOB on day 4 of incubation. An LD50 of 44 ng and 12 ng was estimated
for TCAB and TCAOB, respectively.
Numerous malformations were detected in both hatched
chicks and embryos that died prior to hatching. A direct causal
relationship between TCAB and TCAOB exposure and rump edema was
observed. Edema was detected in embryos and hatched chicks from
eggs treated with 0.01 µg (3.4%) - 1.0 µg (4.2%) of
TCAB and 0.005 µg (2.9%) - 0.05 µg (7.3%) of TCAOB.
The highest incidence of rump edema in treated embryos occurred
in the 0.0075 µg TCAOB/egg (22.5%) and 0.05 µg TCAB/egg
(5.1%) treated fetuses. The percent of embryos with rump edema
ranged from 2.5-5.1% (TCAB) and 2.1-22.5% (TCAOB). Rump edema
was not observed in the control group. All embryos (except 2)
with rump edema died before hatching. The authors concluded that
TCAB and TCAOB are "extremely toxic and potentially teratogenic"
in the chick. However, these compounds appear to be less toxic
than TCDD [Schrankel et al., 1982].
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